Process for the manufacture of 11alpha-hydroxy-6alpha-halogen-pregnenes



3,033,759 PROCESS FDR THE MANUFACTURE GB 110:- HYDRDXY-fiu-HALQGEN-PREGNENES Albert Wettstein, Riehen, and Ernst Vischer, Basel, Switzerland, assignors to Ciba Pharmaceutical Products Inc., Silt, NJ. 7

No Drawing. Filed Feb. 9, 1060, Ser. No. 7,545 Claims priority, application Switzerland Feb. 12, 1959 1 Claim. (Cl. 195-51) ticoid, thymolytic, catabolic, antiandrogenic and antioestrogenic activity. 7

It is known that A*- and A -3-keto-steroids, e.g. A l6cc-methyl-l7azll-dihydroxy pregnadiene 3 :20 dione, can be hydroxylated microbiologically in ll-position.

However, in the case of the fi-halogenated derivatives, the

microbiological introduction of hydroxyl into the ll-position hitherto presented considerable difiiculties.

It has now been found that enzymes of the fungus Aspergilius ochraceus oxidize A -6a-fluoroor -chlorol6-allryl-, in particular A -6a-fluoroor -ChlO1O-l6amethyl-17a 2l-dihydroxy-pregnadiene-3 :20-dione, and A 6a-fiuoroor -chloro-l6-alkyl-, in particular A -6wfluoroor -chloro-16cc-methyl-l7a:2l-dihydroxy pregnene-3 :20- dione to give the corresponding Ila-hydroxy compounds in almost quantitative yield.

The transformation of the above-mentioned starting materials by means of the said fungus,that is with the enzymes obtained therefrom, can be carried out by the working methods known per se for microbiological hydroxylation. The starting materials are generally incubated direct with cultures of the said strains growing under aerobic conditions. These cultures are advantageously moved, i.e. shaken or stirred, and contain assimilable carbon, in particular corn steep liquor or beer wort, and inorganic salts. Thus, natural, synthetic or semisynthetic nutrient solutions can be used. The method which is simplest from the practical point of view is described hereinafter, but it is not intended that the invention be limited by these details:

The organisms are cultivated in apparatus under conditions similar to those known in the manufacture of antibiotics as the so-called deep tank process. The temperature is preferably maintained at 2427 C. and under these conditions the cultures are fully developed after 1-2 days. The starting material is then added under sterile conditions in fine dispersion or solution, for example in methanol, ethanol, acetone, dioxane or propylene glycol, either in a single operation or in stages, and incubation is continued. Finally, the culture filtrate is separated from the mycelium, the mycelium is extracted if required, for example with methanol or acetone, and the concentrated extract is added to the culture filtrate. This is extracted with a suitable organic solvent, such as ethyl acetate, chloroform, ethylene chloride or methylene chloride, the extract is washed with dilute sodium hydrogen carbonate solution and with water and finally it is evaporated. The oxidation product can be isolated from the residue in one of the following Ways: Either the residue is crystallized direct from a suitable solvent, such as, for instance, acetone-petroleum ether, methylene chloride- 3,033,759 Patented May 8, 1962 ether, aqueous methanol, etc., or it is subjected to one of the following preliminary treatments:

Distribution between petroleum ether and methanolwater (:20), the products of the process being enriched in the methanol phase; treatment of 'a solution of the resi due in a suitable solvent such as, for example, methanol, ethanol, acetone or the like withan absorption agent, such as active carbon or aluminum oxide. In this way, the lla-hydroxy-derivatives are obtained in pure form.

The starting materials are new. They can be prepared for example in the following manner: 7

A A 1 935:ZO-diacyloxy-IGu-methyI-pregnadiene of the formula 1 v 1 is oxidized with a peracid, if desired the isomeric mixture of 5 6, l7 20-dioxido-35 20-diacyloxy-l6u-methyl-pregnane obtained is treated withan oxygen-containing mineral acid, the secondary hydroxyl' group in the resulting SazGB-dihydroxy compound is esterified with a sulfonic acid, treatment is then carried out with an alkaline agent, the resulting 5on6 oxido 17cc hydroxy 20 ketone is treated with a hydrogen halide and then with bromine; the 21-b'romide thus formed is treated with a salt of an aliphatic car'boxylic acid of low molecular weight; in the resulting 3: 17a-dihydroxy-2l-acyloxy-5a26a oxido 16ozmethyl-pregnane, before or after oxidizing the 3-hydroxy to the keto group, the 50::6a-OXid0 group is opened to form the 5OL-hYdlOXY-Gfi-flUOIOhYdIlH or Sa-hydroxy-fiflchlorohydrin; the 5a-hydroxyl group is eliminated With formation of the 4:5-double bond; a 6 8-halogeno compound is isomerized to a 6a-halogeno compound; and if desired, a double bond is introduced into the 1:2-position of the resulting A -3:20-dioxo-17a-hydroxy-2l-acyloxyl6oc-m8thYl-6oc-fl11010- or 16a-chloro-pregnene; and/ or in a resulting compound the 2l-acyloxy-group is hydrolized.

The invention is described in the following examples. The temperatures are given in degrees centigrade.

Example 1 4 liters of a nutrient solution containing the following additions to l liter of tap water: 10 grams of crude glucose, 10 grams of distillers solubles, 5 grams of sodium chloride, 1 gram of sodium nitrate and 10 grams of calcium carbonate, are prepared. The pH is adjusted to 7.5 and the solution is sterilized in a shaking vesselfor 30 minutes at 1.1 atmospheres gauge pressure. After cooling, the nutrient solution is inoculated with a culture of Aspergillus ochmceus (Ciba 924), after which the vessel is shaken at a temperature of 25 C. After 2 days, a solution of 1 gram of A -6a-fluoro-l6a-methyl-170::21- dihydroxy-pregnene-3:20-dione-2l-acetate in 25 cc. of methanol is added to the well-developed culture under sterile conditions and shaking is continued for a further 48 hours at the same temperature. The mycelium is then separated, suspended for one hour in 800cc. of Warm acetone and once more filtered off. The acetone filtrate is thereupon strongly concentrated in vacuo and then combined with the culture filtrate. The combined filtrates are extracted three times with 0.8 liter of acetic ester in each case and the extracts are then washed with a 2% solution of sodium hydrogen carbonate and with water, dried over sodium sulfate, filtered and evaporated to dryness in vacuo at a temperature of 3035 C. 1.4 grams of an oily residue are obtained, the steroid constituent of t f :5. which consists according to evaluation by paper chromatography, almost exclusively of A -6ot-fiuoro-16ot-methy1- 11a:17a:2l-trihydroXy-pregnene-3:ZO-dione. This 'residuais. dissolved in sun, of methanol and-100 cc. oi

petroleum ether aud 20 cc. of Water are added to the solu-.

tion; and the mixture is shaken. The methanol phase is thereupon separated ofi, shaken up twice more with 3 5 cc. of petroleum ether; in each case and after the petroleum etherphase has been separated oil isevaporated at a temperature of 3035 in vacuo. "980 mg. of a partially crystalline jresidueare obtained. By recrystallization from aniacetone-ether mixture A -a-fiuororl6m-methyl- 11cc: 1 7 x 22-l trihydrexy-pregnene :ZO-dione is in purefform. r

i In similar-manner there isob tained from A 6-flu0rol6u-methyl-l7uz2l dihydroky-pregnadiene- 3 z20-dione- ZI-acetate, by crystallization of the crude oxidation prod.

Example 3 V If a nutrient solution containing 2.6 grams of ammonium tartrate, 2.6 grams of tartaric acid, 0.4 gram of potassium carbonate, 0.4 gram of ammonium phosphate, 0.27 gram of magnesium carbonate, 0.17 gram of ammonium sulfate and 50 grams of crude glucose to l-liter of tap Water is'employed, excellent transformation of obtained net obtained fromv an acetone-ether mixture, A -6aflLlOIOJGoc-DlethYI-lla:171x221 trihydroxy pregnadiene! 3:20-dione in colorless crystals. a

v V Example 2 A 4-liter culture of Aspergillus ochraceus (Ciba 924) is prepared as described in Example 1 and a solution of 1 'gram of A r60L-vflUOIO-160L-1I16thY1-170tI21-dlhYdfOXY- pregnene-BzZO-dione in 2 5 cc. of methanol is added there to under sterile conditions. After incubation and working up as indicated in Example 'l, homogeneous A 60;- fluoro 16a methyl 11a: 170::21 trihydroxy' pregnene- .-.3':20-dione is obtained. v

20 enzyme of the fungus Aspergillus ochraceus.

References Citedin the. file of this patent UNITED STATESPATENTS 2,649,402 Murray et al Aug. 18, 1953 2,802,775 Dulaney et a1. Aug. 13, 1957 2,905,593 Dulaneyet a1. -4- Sept. 22, 1959 so I: 

